Apparatus and methods for parallel processing of micro-volume liquid reactions

ABSTRACT

Disclosed herein are apparatuses and methods for conducting multiple simultaneous micro-volume chemical and biochemical reactions in an array format. In one embodiment, the format comprises an array of microholes in a substrate. Besides serving as an ordered array of sample chambers allowing the performance of multiple parallel reactions, the arrays can be used for reagent storage and transfer, library display, reagent synthesis, assembly of multiple identical reactions, dilution and desalting. Use of the arrays facilitates optical analysis of reactions, and allows optical analysis to be conducted in real time. Included within the invention are kits comprising a microhole apparatus and a reaction component of the method(s) to be carried out in the apparatus.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/935455, filed Aug. 22, 2001 and published as U.S. Publication No.20020151040 on Oct. 17, 2002, which is a continuation in part of U.S.patent application Ser. No. 09/789,899, filed Feb. 20, 2001, whichclaims priority to U.S. Provisional application Ser. No. 60/229,357,filed Feb. 18, 2000, the disclosures of which are incorporated herein byreference.

Statement of Rights to Inventions Made Under Federally SponsoredResearch

Not applicable.

TECHNICAL FIELD AND BACKGROUND ART

This invention is related to devices and processes for carrying outmultiple simultaneous micro-reactions in liquid samples.

BACKGROUND

Reactions that are conducted in solution such as, for example, chemical,biological, biochemical and molecular biological reactions, arefrequently carried out within a chamber or other container. Suchchambers, or reaction vessels, are commonly made of glass or plastic andinclude, for example, test tubes, microcentrifuge tubes, capillary tubesand microtiter plates. Reaction chambers currently in use are notamenable for use with volumes below one microliter, due to problems suchas large head volumes in the reaction chamber leading to evaporativelosses of the reaction solution, and difficulty in adding and removingreaction mixtures from the reaction chamber.

Many types of biochemical reactions, for example, nucleic acidamplification, require temperature cycling. Many reaction chambermaterials are poor thermal conductors, thus there are time lagsassociated with changing the temperature of the reaction vessel andequilibration of a temperature change throughout the sample volume. Suchlags in temperature change and temperature equilibration lead to longercycle times, non-uniform reaction conditions within a single reaction,and lack of reproducibility among multiple reactions, both simultaneousand sequential.

It is often necessary to carry out a series of experiments on a set ofidentical samples. Usually this set of samples must be seriallyduplicated, either manually or by means of robotic liquid deliverysystems. These processes can be slow, as they depend on the total numberof samples to be duplicated and, if applicable, the speed of the robot.

Efforts to address the aforementioned problems have included the use ofrobotics and the use of capillary thermal cyclers, e.g., the LightCycler.RTM. (Idaho Technologies). See Wittwer et al. (1997a)BioTechniques 22:130-138, and Wittwer et al. (1997b) BioTechniques22:176-181. However, such methods and apparatuses still require samplevolumes of several microliters, involve difficult liquid handlingprocedures such as loading and emptying capillaries, and can involvedetection problems associated with capillary geometry and spacing.

Microarrays comprising an ordered array of biological molecules (e.g.,peptides, oligonucleotides) on a solid surface are known. See, forexample, U.S. Pat. Nos. 5,445,934; 5,510,270; 5,605,662; 5,632,957;5,744,101; 5,807,522 5,929,208 and PCT publication WO 99/19510. Whilethese are useful for analyzing multiple molecules under identical,conditions (e.g., hybridizing a plurality of different oligonucleotidesequences with a single probe or probe mixture), such a “chip” cannot beused for analysis of multiple samples under multiple experimentalconditions. Furthermore, such arrays are limited to analysis ofmolecules which can either be synthesized on the array substrate orcovalently attached to the substrate in an ordered array. In addition,molecules tethered to an array react with slower kinetics than domolecules in solution, and are sterically hindered in their interactionsresulting in altered reaction kinetics. Additionally, for arrays ofproteins and peptides, surface interactions affect the naturalconformation of proteins under investigation (MacBeath and Schreiber,Science, vol 289, pp. 1760-1763). 5 WO 99/34920 discloses a system andmethod for analyzing a plurality of liquid samples, the systemcomprising a platen having two substantially parallel planar surfacesand a plurality of through-holes dimensioned so as to maintain a liquidsample in each through-hole by means of surface tension. WO 00/56456discloses a method for holding samples for analysis and an apparatusthereof includes a testing plate with a pair of opposing surfaces and aplurality of holes. WO 99/47922 discloses vascularized perfusedmicrotissue/micro-organ arrays. U.S. Pat. No.5,290,705 discloses aspecimen support for optical observation or analysis, the supportcomprising a disc-like member composed of a rigid material and having atleast one unobstructed hole extending therethrough.

Polynucleotides may be sheared through transfer methods such aspipetting. One method for reducing polynucleotide shear is to use pipettips with the tip ends cut off for polynucleotide transfer. Othermethods for reducing polynucleotide shear have been described in U.S.Pat. Nos. 6,147,198; 4,861,448; 5,599,664; 5,888,723; and 5,840,862.

There is a continued need for apparatuses and methods suitable formicrovolume liquid reactions. There is also a need for improved methodsof transferring polynucleotides.

All references cited herein are hereby incorporated by reference intheir entirety.

DISCLOSURE OF THE INVENTION

Disclosed herein are apparatuses for containing multiple micro-volumesamples and conducting multiple simultaneous micro-volume chemical andbiochemical reactions in an array format, methods utilizing theapparatuses, and kits containing the apparatuses.

The embodiments of the invention include, but are not limited to, thefollowing.

An apparatus for containing multiple micro-volume liquid samplescomprising a substrate, wherein the substrate defines a plurality ofsample chambers, wherein each sample chamber: (a) extends through thesubstrate, (b) comprises one or more walls and an opening at each end,and (c) holds a sample such that the sample is in the form of a thinfilm such that a liquid sample present in one sample chamber does notintermix with a liquid sample present in another sample chamber; andwherein the sample chamber has a height to width ratio of less than 1:1, wherein the height of the sample chamber is measured from one face ofthe substrate to the other. The apparatus may comprise at least onecomponent of a reaction to be carried out in the apparatus. In oneembodiment the component is a reagent used in a nucleotide sequencingreaction. In another embodiment the component is one used in ahybridization reaction. In another embodiment, the apparatus issubstantially free from contaminating amplifiable polynucleotides.

In another aspect of the invention, an apparatus for containing multiplemicro-volume liquid samples comprising a substrate is provided, whereinthe substrate defines a plurality of sample chambers, wherein eachsample chamber: (a) extends through the substrate, (b) comprises one ormore walls and an opening at each end, and (c) holds a sample such thatthe sample is retained in the apparatus through surface tension and suchthat a liquid sample present in one sample chamber does not intermixwith a liquid sample present in another sample chamber; wherein theapparatus is substantially free of contaminating amplifiablepolynucleotides; and wherein the apparatus comprises at least onereagent used in a polynucleotide amplification reaction to be carriedout in the apparatus. In one embodiment, the apparatus comprises atleast two reagents used in a polynucleotide amplification reaction to becarried out in the apparatus. In a preferred embodiment, the samplechamber has a height to width ratio of about 1: 1, wherein the height ofthe sample chamber is measured from one face of the substrate to theother. The polynucleotide amplification reaction may be, for example, apolymerase chain reaction, a ligase chain reaction, or a rolling circleamplification reaction.

For apparatuses disclosed herein, the reaction component(s) mayoptionally be affixed to the solid substrate. In some embodiments thereaction component is affixed to the solid substrate by drying.

In a preferred embodiment, the substrate comprises hydrophobic regions;the hydrophobic regions are located on the substrate such that a liquidsample present in one sample chamber does not intermix with a liquidsample present in another sample chamber. In an embodiment of theapparatus, the hydrophobic regions are located on the upper and lowerfaces of the substrate such that the openings of at least one samplechamber from at least one adjacent sample chamber by a hydrophobicregion. The hydrophobic regions may also be located on the walls of thesample chambers. In some embodiments the hydrophobic region forms anannular ring along the wall of the sample chamber. In some embodimentsthe apparatus comprises two or more hydrophobic regions, each forming anannular ring along the wall of the sample chamber, and the hydrophobicregions define one or more annular non-hydrophobics rings therebetween.

In another embodiment of the apparatus the substrate can comprise anupper face and a lower face. A futher refinement of this embodiment iswherein the through axes of the sample chambers are perpendicular toboth faces of the substrate. The sample chamber may also have the shapeof, for example, a right circular cylinder or a right polygonal prism.

Other embodiments of the invention are methods that are carried out in amicrohole apparatus. These methods include the following:

A method for simultaneously conducting a plurality of micro-volumereactions, the method comprising: (a) introducing a plurality of liquidsamples into the sample chambers of a microhole apparatus, wherein thesamples contain necessary reaction components; and (b) placing theapparatus into an environment favorable to the reaction; wherein themicrohole apparatus comprises a substrate, wherein the substrate definesa plurality of sample chambers, wherein each sample chamber: (i) extendsthrough the substrate; (ii) comprises one or more walls and an openingat each end; and holds a sample such that the sample is in the form of athin film such that a liquid sample present in one sample chamber doesnot intermix with a liquid sample present in another sample chamber; andwherein the sample chamber has a height to width ratio of less than 1:1,wherein the height of the sample chamber is measured from one face ofthe substrate to the other.

The environment can be one to prevent evaporation, such as a hydrophobicmedium or a humidified chamber. In some embodiments the apparatus issubstantially free of contaminating ampliflable polynucleotides. In someembodiments the reactions can be ligation reactions, primer extensionreactions, nucleotide sequencing reactions, restriction endonucleasedigestions, oligonucleotide synthesis reactions, hybridizationreactions, and biological interactions. The biological interactions canbe avidin-biotin interactions, streptavidin-biotin interactions,antigen-antibody interactions, hapten-antibody interactions andligand-receptor interactions. In some embodiments the reaction componentis affixed to the substrate. In some embodiments the results of thereactions are monitored. Monitoring can be by a number of methods,including optical monitoring, mass spectrometry and electrophoresis.Monitoring can be of the progress of the reactions during the course ofthe reactions. In some of the methods of the invention one or more ofthe reactions are supplemented with one or more reagents during thecourse of the reaction.

In another method of the invention utilizing a microhole apparatus isone for adding a component to a microvolume reaction. The methodcomprises the steps of: A method for adding a component to amicro-volume reaction, wherein the method comprises the steps of: (a)providing a first apparatus comprising a first sample chamber containinga reaction mixture; (b) providing a second apparatus comprising a secondsample chamber containing the component; and (c) bringing theapparatuses into proximity such that liquid contact is establishedbetween the first sample chamber and the second sample chamber; whereineach apparatus comprises a substrate, wherein the substrate defines aplurality of sample chambers, wherein each sample chamber: (i) extendsthrough the substrate; (ii) comprises one or more walls and an openingat each end; and (iii) holds a sample such that the sample is in theform of a thin film such that a liquid sample present in one samplechamber does not intermix with a liquid sample present in another samplechamber; and wherein the sample chamber has a height to width ratio ofless than 1:1, wherein the height of the sample chamber is measured fromone face of the substrate to the other. In another embodiment of themethod multiple components are added to a reaction, by providingadditional apparatuses, wherein each of the components is present in asample chamber of an apparatus. Another embodiment comprisessimultaneously adding a component to a plurality of micro-volumereactions wherein, in the method described above, the apparatuses arebrought into proximity such that liquid contact is established betweencorresponding sample chambers of the apparatuses. In a preferredembodiment the component is a nucleic acid.

A method for adding a nucleic acid to a micro-volume reaction isprovided, wherein the method comprises the steps of: (a) providing afirst apparatus comprising a first sample chamber containing a reactionmixture; (b) providing a second apparatus comprising a second samplechamber containing the nucleic acid; and (c) bringing the apparatusesinto proximity such that liquid contact is established between the firstsample chamber and the second sample chamber; wherein each apparatuscomprises a substrate, wherein the substrate defines a plurality ofsample chambers, wherein each sample chamber: (i) extends through thesubstrate; (ii) comprises one or more walls and an opening at each end;and (iii) holds a sample such that the sample is retained in theapparatus through surface tension and such that a liquid sample presentin one sample chamber does not intermix with a liquid sample present inanother sample chamber.

A method for introducing a liquid sample into a sample chamber isprovided, wherein the method comprises the steps of: (a) contacting anapparatus with a liquid solution; and (b) removing the apparatus fromthe solution; wherein the apparatus comprises a substrate, wherein thesubstrate defines a plurality of sample chambers, wherein each samplechamber: (i) extends through the substrate; (ii) comprises one or morewalls and an opening at each end; and (iii) holds a sample such that thesample is in the form of a thin film such that a liquid sample presentin one sample chamber does not intermix with a liquid sample present inanother sample chamber; and wherein the sample chamber has a height towidth ratio of less than 1: 1, wherein the height of the sample chamberis measured from one face of the substrate to the other.

In another embodiment of the invention, a method for introducing aliquid sample comprising a nucleic acid into a sample chamber isprovided, wherein the method comprises the steps of: (a) contacting anapparatus with a liquid solution comprising a nucleic acid; and (b)removing the apparatus from the solution; wherein the apparatuscomprises a substrate, wherein the substrate defines a plurality ofsample chambers, wherein each sample chamber: (i) extends through thesubstrate; (ii) comprises one or more walls and an opening at each end;and (iii) holds a sample such that the sample is retained in theapparatus through surface tension and such that a liquid sample presentin one sample chamber does not intermix with a liquid sample present inanother sample chamber.

In another embodiment of the invention, a method for diluting a solutionis provided, wherein the method comprises the steps of: (a) providing afirst apparatus comprising a first sample chamber containing thesolution (b) providing a second apparatus comprising a second samplechamber containing a diluent; and (c) bringing the apparatuses intoproximity such that liquid contact is established between the firstsample chamber and the second sample chamber; wherein each of theapparatuses comprises a substrate, wherein the substrate defines aplurality of sample chambers, wherein each sample chamber: (i) extendsthrough the substrate; (ii) comprises one or more walls and an openingat each end; and (iii) holds a sample such that the sample is in theform of a thin film such that a liquid sample present in one samplechamber does not intermix with a liquid sample present in another samplechamber; and wherein the sample chamber has a height to width ratio ofless than 1:1, wherein the height of the sample chamber is measured fromone face of the substrate to the other.

In another embodiment of the invention, a method for diluting a solutioncomprising a nucleic acid is provided, wherein the method comprises thesteps of: (a) providing a first apparatus comprising a first samplechamber containing the solution which comprises a nucleic acid; (b)providing a second apparatus comprising a second sample chambercontaining a diluent; and (c) bringing the apparatuses into proximitysuch that liquid contact is established between the first sample chamberand the second sample chamber; wherein each of the apparatuses comprisesa substrate, wherein the substrate defines a plurality of samplechambers, wherein each sample chamber: (i) extends through thesubstrate; (ii) comprises one or more walls and an opening at each end;and (iii) holds a sample such that the sample is retained in theapparatus through surface tension and such that a liquid sample presentin one sample chamber does not intermix with a liquid sample present inanother sample chamber.

In preferred embodiments for the methods described above a plurality ofsolutions may be simultaneously diluted wherein the apparatuses arebrought into proximity such that liquid contact is established betweencorresponding sample chambers of the apparatuses. In another preferredembodiment, steps (b) through (c) are repeated one or more times using anew second apparatus containing fresh solvent at each repetition of step(b).

In another embodiment of the invention, a method for selective retentionof a molecule in a first sample chamber is provided, wherein the methodcomprises the steps of: (a) providing a first apparatus, wherein thefirst sample chamber contains a solution comprising the molecule and oneor more additional solute molecules of higher diffusibility; (b)providing a second apparatus comprising a second sample chambercontaining a solvent; (c) bringing the apparatuses into proximity suchthat liquid contact is established between the first sample chamber andthe second sample chamber; and (d) removing the apparatuses fromproximity; wherein each of the apparatuses comprises a substrate,wherein the substrate defines a plurality of sample chambers, whereineach sample chamber: (i) extends through the substrate; (ii) comprisesone or more walls and an opening at each end; and (iii) holds a samplesuch that the sample is in the form of a thin film such that a liquidsample present in one sample chamber does not intermix with a liquidsample present in another sample chamber; and wherein the sample chamberhas a height to width ratio of less than 1: 1, wherein the height of thesample chamber is measured from one face of the substrate to the other.

A method for selective retention of a nucleic acid in a first samplechamber is provided, wherein the method comprises the steps of: (a)providing a first apparatus, wherein the first sample chamber contains asolution comprising the nucleic acid and one or more additional solutemolecules of higher diffusibility; (b) providing a second apparatuscomprising a second sample chamber containing a solvent; (c) bringingthe apparatuses into proximity such that liquid contact is establishedbetween the first sample chamber and the second sample chamber; and (d)removing the apparatuses from proximity; wherein each of the apparatusescomprises a substrate, wherein the substrate defines a plurality ofsample chambers, wherein each sample chamber: (i) extends through thesubstrate; (ii) comprises one or more walls and an opening at each end;and (iii) holds a sample such that the sample is retained in theapparatus through surface tension and such that a liquid sample presentin one sample chamber does not intermix with a liquid sample present inanother sample chamber.

In a preferred embodiment of these methods, the method is used fordesalting a solution. In another preferred embodiment, steps (b) through(d) are repeated one or more times using a new second apparatuscontaining fresh solvent at each repetition of step (b). In anotherpreferred embodiment, a plurality of solutions may be simultaneouslydesalted, wherein the apparatuses are brought into proximity such thatliquid contact is established between corresponding sample chambers ofthe apparatuses.

A method for parallel electrophoretic analysis of a plurality ofmicro-volume reactions is provided, wherein the method comprises: (a)conducting the reactions in a microhole apparatus; (b) placing theapparatus in contact with an electrophoresis medium; and (c) conductingelectrophoresis; wherein the apparatus comprises a substrate, whereinthe substrate defines a plurality of sample chambers, wherein eachsample chamber: (i) extends through the substrate; (ii) comprises one ormore walls and an opening at each end; and (iii) holds a sample suchthat the sample is retained in the apparatus through surface tension andsuch that a liquid sample present in one sample chamber does notintermix with a liquid sample present in another sample chamber. Theapparatus can be placed with one face in contact with theelectrophoresis medium. In another embodiment the electrophoresis mediumis contained within the sample chambers of one or more additionalapparatuses. In other embodiments, in the additional apparatuses thecorresponding sample chambers are aligned.

Another method for use in the invention comprises a method for preparinga plurality of samples for mass spectrometric analysis, wherein thesamples are placed in an apparatus that comprises a substrate, whereinthe substrate defines a plurality of sample chambers, wherein eachsample chamber: (a) extends through the substrate; (b) comprises one ormore walls and an opening at each end; and (c) holds a sample such thatthe sample is retained in the apparatus through surface tension and suchthat a liquid sample present in one sample chamber does not intermixwith a liquid sample present in another sample chamber. The analysis canbe conducted by matrix assisted laser desorption ionizationtime-of-flight (MALDI-TOF) spectrometry. These methods can be used todetect a genetic polymorphism, for example, a single nucleotidepolymorphism (SNP). Detection may consist of, for example, massdifferences due to a single base primer extension.

A method for mixing a plurality of micro-volume samples is provided, themethod comprising: (a) providing a first microhole apparatus comprisinga substrate, wherein the substrate defines a plurality of samplechambers, wherein each sample chamber: (i) extends through thesubstrate;(ii) comprises one or more walls and an opening at each end;and (iii) holds a sample such that the sample is retained in theapparatus through surface tension and such that a liquid sample presentin one sample chamber does not intermix with a liquid sample present inanother sample chamber; (b) providing a second microhole apparatuscomprising a substrate, wherein the substrate defines a plurality ofsample chambers, wherein each sample chamber:(i) extends through thesubstrate;(ii) comprises one or more walls and an opening at each end;and (iii) holds a sample such that the sample is retained in theapparatus through surface tension and such that a liquid sample presentin one sample chamber does not intermix with a liquid sample present inanother sample chamber; and (c) bringing the apparatuses into proximitysuch that liquid contact is established between more than one samplechamber from the first apparatus and a sample chamber in the secondapparatus. In one embodiment, the holes in the first apparatus may besmaller than the holes in the second, allowing more than one hole fromthe first apparatus to simultaneously contact a single hole in thesecond apparatus.

A method for simultaneously conducting a plurality of micro-volumepolynucleotide amplification reactions is provided, the methodcomprising: (a) introducing a plurality of liquid samples into thesample chambers of a microhole apparatus, wherein the samples containnecessary polynucleotide amplification reaction components; and (b)placing the apparatus into an environment favorable to thepolynucleotide amplification reaction; wherein the microhole apparatuscomprises a substrate, wherein the substrate defines a plurality ofsample chambers, wherein each sample chamber: (i) extends through thesubstrate; (ii) comprises one or more walls and an opening at each end;and (iii) holds a sample such that the sample is retained in theapparatus through surface tension and such that a liquid sample presentin one sample chamber does not intermix with a liquid sample present inanother sample chamber; and wherein the apparatus is substantially freeof contaminating amplifiable polynucleotides.In one embodiment, theenvironment is selected from the group consisting of a hydrophobicmedium and a humidified chamber. In another embodiment, thepolynucleotide amplification reaction is a polymerase chain reaction. Inother embodiments, the polynucleotide amplification reaction is a ligasechain reaction or a rolling circle amplification reaction. In someembodiments, the results of the reactions are monitored. The results maybe monitored by, for example, optical monitoring, mass spectrometry andelectrophoresis. In another embodiment, the progress of the reactionsare monitored during the course of the reactions. In yet anotherembodiment, one or more of the reactions are supplemented with one ormore reagents during the course of the reaction. In a preferredembodiment, a reagent is affixed to the substrate. In another preferredembodiment, the analysis is used to detect a genetic polymorphism. Inyet another preferred embodiment, the analysis is used to analyze geneexpression levels.

Still other embodiments of the invention are kits containing themicrohole apparatuses described Supra, for the methods of the invention.These include the following.

A kit comprising an apparatus for containing multiple micro-volumeliquid samples comprising a substrate, wherein the substrate defines aplurality of sample chambers, wherein each sample chamber: (a) extendsthrough the substrate, (b) comprises one or more walls and an opening ateach end, and (c) holds a sample such that the sample is in the form ofa thin film such that a liquid sample present in one sample chamber doesnot intermix with a liquid sample present in another sample chamber; andwherein the sample chamber has a height to width ratio of less than 1:1, wherein the height of the sample chamber is measured from one face ofthe substrate to the other, and further comprising a reaction componentpackaged in a suitable container. The reaction component may be areagent for performing a reaction selected from the group consisting of,for example, ligation reactions, primer extension reactions, nucleotidesequencing reactions, restriction endonuclease digestions,oligonucleotide synthesis, hybridization reactions and biologicalinteractions.

A kit comprising an apparatus for containing multiple micro-volumeliquid samples comprising a substrate is provided, wherein the substratedefines a plurality of sample chambers, wherein each sample chamber: (a)extends through the substrate, (b) comprises one or more walls and anopening at each end, and (c) holds a sample such that the sample isretained in the apparatus through surface tension and such that a liquidsample present in one sample chamber does not intermix with a liquidsample present in another sample chamber; wherein the apparatus issubstantially free of contaminating amplifiable polynucleotides, andfurther comprising a polynucleotide amplification reaction componentpackaged in a suitable container.

In one embodiment of the above described kits, the reaction componentmay be affixed to the substrate. In another embodiment, the kit mayfurther comprise a hydrophobic substance to be used with the apparatus.The hydrophobic substance can be, for example, a hydrophobic fluidpackaged in a suitable container and/or a hydrophobic cover. The kit mayalso further comprise a chamber for maintaining the appropriateenvironmental conditions for a reaction to be carried out in theapparatus. The kit may also further comprise an apparatus for loadingthe samples into the sample chambers.

As will be apparent to one of skill in the art, any method or techniquewhich requires parallel processing, display and/or storage of multiplemicro-volume samples will be facilitated by the use of the apparatusesdisclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of one embodiment of the claimed apparatus,in the form of a microhole array.

FIG. 2 is a side view (cutaway) through one row of an exemplaryapparatus.

FIGS. 3A and 3B show side (cutaway) views of a single exemplary samplechamber.

FIGS. 4A and 4B show side (cutaway) views of exemplary sample chamberscontaining alternating hydrophobic and hydrophilic regions.

FIGS. 5A, 5B and 5C show simultaneous loading of multiple individualsamples into discrete sample chambers by contacting an exemplaryapparatus with an arrangement of liquid samples on a hydrophobicsurface.

FIG. 6A, 6B and 6C are top views of exemplary apparatuses that can beused for gel loading.

FIG. 7 is a top view of one embodiment of the claimed apparatus, whereinthe apparatus is taped with aluminum tape.

MODES FOR CARRYING OUT THE INVENTION

General Methods:

The practice of the invention employs, unless otherwise indicated,conventional techniques in photolithography, chemical etching, generalmachining, microfluidics, organic chemistry, biochemistry,oligonucleotide synthesis and modification, nucleic acid hybridization,molecular biology, microbiology, genetic analysis, recombinant DNA, andrelated fields as are within the skill of the art. These techniques aredescribed in the references cited herein and are fully explained in theliterature. See, for example, Maniatis, Fritsch & Sambrook, MOLECULARCLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press(1982); Sambrook, Fritsch & Maniatis, MOLECULAR CLONING: A LABORATORYMANUAL, Second Edition, Cold Spring Harbor Laboratory Press (1989);Ausubel, et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley &Sons (1987 and annual updates); Gait (ed.), OLIGONUCLEOTIDE SYNTHESIS: APRACTICAL APPROACH, IRL Press (1984); Eckstein (ed.), OLIGONUCLEOTIDESAND ANALOGUES: A PRACTICAL APPROACH, IRL Press (1991); Birren et al.(eds.) GENOME ANALYSIS: A LABORATORY MANUAL, Cold Spring HarborLaboratory Press, 1999.

The disclosures of all publications and patents cited herein are herebyincorporated by reference in their entirety.

Definitions

The terms “plate” and “substrate” denote the solid portion of anapparatus.

A characteristic of a “thin-film” sample, as disclosed herein, is that asample is contained in a sample hole and remains therein through theaction of surface tension and/or adhesion to the inner wall of the hole.Preferably, a thin film is a liquid sample in which the diffusion timeis no more than about four-fold greater, more preferably no more thanabout three-fold greater, more preferably no more than about two-foldgreater, more preferably no more than about one-fold greater in onedimension that in any other dimension. Preferably, the temperatureconductance characteristics of a thin film sample are no more than aboutfour-fold greater, more preferably no more than about three-foldgreater, more preferably no more than about two-fold greater, even morepreferably no more than about one-fold greater in one dimension that inany other dimension. Most preferably, a thin film will have hydrodynamicand thermal properties equivalent to a solution contained in a rightcircular cylinder having a depth:diameter ratio of about 4:1, or morepreferably about 3:1 or less, about 2:1 or less, more preferably about1:1, even more preferably less than 1:1. Methods for measuringhydrodynamic properties, diffusion time and thermal conductancecharacteristics are well-known to those of skill in the art.

Two or more holes in an apparatus are denoted “corresponding holes” ifthey occupy the same relative position on two or more differentapparatuses such that, if the apparatuses are aligned face-to-face, theholes communicate with one another. “Polynucleotide”, “oligonucleotide”,and “nucleic acid”, are used interchangeably herein to refer to polymersof nucleotides of any length, and includes natural, synthetic, andmodified nucleic acids. “Substantially free of contaminating amplifiablepolynucleotides”, as used herein, is meant to indicate an apparatuswhich is substantially free from contaminating polynucleotides, such asDNA or RNA, which may interfere with the analysis. Such an apparatus issuitable for use in performing assays such as, for example,amplification reactions, e.g. PCR reactions, in which contaminatingamplifiable polynucleotides may coamplify along with the desiredamplification product(s), thus interfering with the analysis.

Apparatuses

Disclosed herein are apparatuses and methods for simultaneous parallelprocessing, display and/or storage of a plurality of micro-volume liquidsamples, wherein an apparatus comprises a substrate containing aplurality of micro-sample chambers. In one embodiment, the samplechambers are micro-through-holes, such that the apparatus comprises anarray of micro-through-holes in a substrate. The apparatus can have anyshape consonant with the purposes for which it is used. In oneembodiment, the apparatus is rectangular; however, triangular, circularand ovoid shapes, among others, are also useful. An array of microholes,as disclosed herein, can be used, for example, as a micro-volume sampleholder and/or to conduct multiple parallel reactions.

Microholes can be placed in any arrangement within a substrate that issuitable for the experimental purpose of the apparatus. In oneembodiment, holes are arranged in rows and columns on a rectangularsubstrate. The size and/or shape of an apparatus can vary, and isdesigned with the particular experimental use of the apparatus in mind.For example, if the apparatus is to be used for gel loading or if theproducts of a reaction conducted in the apparatus are to be analyzed bygel electrophoresis (see infra), the size and shape of the apparatus canbe designed to match that of a gel electrophoresis apparatus or samplecomb.

An exemplary embodiment of the apparatus is described with reference toFIGS. 1 and 2, wherein the apparatus 1 comprises an array ofmicro-through-holes 5 contained in a substrate 6, such as a plate,wafer, film or slide, such substrate in one embodiment beingsubstantially thin and planar, and having an upper surface 7 and a lowersurface 8. In one embodiment, one or more of the surfaces of thesubstrate are rendered hydrophobic so that liquid reaction mixturescontained in the micro-through-holes will, by force of surface tensionand adhesion, remain fixed therein. In another embodiment, the substrateis flexible. In yet another embodiment, the substrate is a curved plane.An exemplary use of the latter embodiments is to bend the substrate intoa cylinder and place a rotating optical scanner inside the cylinder tomonitor the reactions in the microholes.

Any size and/or shape of the sample chamber, that is consistent with theretention of liquid therein through surface tension and is commensuratewith the use of the apparatus, can be chosen. In. one preferredembodiment, the sample chambers are in the shape of a right polygonalprism, for example, a right rectangular prism. Although, in a preferredembodiment, the sample chambers are in the shape of right circularcylinders with parallel walls, it is clear that the walls of a samplechamber could be convex (i.e., bowed inward) or concave (i.e., bulgedoutward). Additional sample chamber configurations will be apparent tothose of skill in the art and, indeed, any shape of sample chamberconsistent with the retention of liquid therein through surface tensionis useful. In one embodiment, the height of the hole is greater thanabout four times the width. In other embodiments, the height of the holeis less than or equal to about four times the width, less than or equalto about three times the width, less than or equal to about 2.5 timesthe width, less than or equal to about two times the width, equal toabout one times the width, less than one times the width, less than orequal to about 0.5 times the width. In another preferred embodiment, theheight is equal to or less than the width. In a preferred embodiment,the height of the hole is about 1 times the width. In this embodiment,the sample chamber is a microhole having an aspect ratio with a width 11roughly equal to depth 12. See FIG. 3A for the case of a cylindricalsample chamber. In additional embodiments, the width 11 is greater thanthe depth 12. See FIG. 3B, again directed to the exemplary case in whichthe sample chamber is a cylinder. Thus, in a preferred embodiment, thediffusion time across the height is equal to or less than the diffusiontime across the width. For a hole having the shape of a right circularcylinder, the height:width ratio can also be expressed as the ratio ofdepth to diameter.

The size of the holes is commensurate with the reaction volume and canbe varied by varying the width (or diameter) of the hole and/or thethickness of the substrate (which effectively varies the height or depthof the hole). Thus, the volume of a reaction which can be contained in ahole is a function of the height of the hole and the width of the hole.However, a hole can be loaded such that the liquid extends beyond thephysical boundaries of the hole; in some cases this will be facilitatedif the surface of the substrate surrounding the openings of the holescomprises a hydrophobic material; in other cases, it will beaccomplished by surface tension. In this fashion, a volume of liquidwhich is greater than the volume of the hole can be accommodated by asample chamber. Conversely, a hole can be loaded with a volume of liquidthat is less than the volume of the hole, such that the liquid sampleforms a biconcave film. Thus the shape of the sample can range from abiconvex disc through a flat disc to a biconcave disc. Accordingly,sample volumes of less than about 10000 nl, preferably less than about1000 nl, preferably less than about 500 nl, preferably less than about100 nl, more preferably less than about 250 nl, more preferably lessthan about 100 nl, more preferably, less than about 50 nl can bereliably achieved. In one embodiment, sample volumes as low as 5 nl areused. Thus, sample volumes contemplated range from about 1 nl to about10000 nl.

Each sample chamber can contain an individual sample, or a samplechamber can contain multiple samples separated by hydrophobic regionsalong the wall of the sample chamber. Thus, in one embodiment, theentire inner wall of a sample chamber is hydrophilic and the samplechamber contains a single sample. In another embodiment, the inner wallof a sample chamber is hydrophobic. In another embodiment, hydrophobicregions are located on the walls of the sample chambers. In a furtherembodiment, a hydrophobic region forms an annular ring along the wall ofthe sample chamber. Such a hydrophobic ring can be used, for example, todivide a sample chamber into two regions. Dual-region sample chamberscan be used, for example, to temporarily isolate different reactioncomponents prior to mixing by, for example, physical agitation,insertion and optionally movement of a probe and/or heating (e.g.,interior laser heating). Such a configuration is useful in the practiceof methods such as, for example, hot-start PCR. Additional applicationsof such a configuration will be apparent to those of skill in the art.

In accord with this embodiment, FIG. 4A shows a schematic diagram of anexample of a microhole sample chamber containing annular hydrophobicregion 21 along its wall, separating hydrophilic regions 22 and 23. Inadditional embodiments, the wall of a sample chamber comprises two ormore hydrophobic regions, each forming an annular ring along the wall ofthe sample chamber, thereby defining a plurality of annularnon-hydrophobic rings. One example is diagrammed in FIG. 4B, which showsa schematic diagram of an exemplary microhole sample chamber containingannular hydrophobic regions 25 and 26 along its wall, interspersed withannular hydrophilic regions 27, 28 and 29.

Hydrophobic and/or hydrophilic regions along the wall of a samplechamber need not form a continuous 360.degree. ring, nor need they be inthe shape of an annulus or portion thereof. Arcs or spots of hydrophilicand/or hydrophobic regions can be present, as required by the particularuse of the apparatus. For example, a hydrophobic annular ring,separating two aqueous samples, can be interrupted by a smallhydrophilic arc, the presence of which facilitates eventual mixing ofthe samples. In another example, a small hydrophilic region can bepresent on the wall of the sample chamber such that an aqueous samplecan be concentrated and optionally dehydrated onto the small hydrophilicregion. This can facilitate mass spectrometric analysis of a sample, byconcentrating the sample into a small target for the laser that is usedto launch the sample for mass spectrometry.

Apparatuses comprising holes with hydrophilic and hydrophobic regionslocated on the walls of the sample chamber may be constructed, forexample, by laminating plates comprising different materials togetherwith, for example, epoxy. For example, a titanium plate with chemicallyetched holes can be laminated on both sides to plastic plates alsoetched with corresponding holes, yielding an apparatus with microholeshaving an annular hydrophilic ring surrounded by annular rings ofhydrophobic regions.

In one embodiment, the apparatus is a plate with holes passing throughin a direction perpendicular to at least one face of the plate. Inanother embodiment, the faces are parallel to each other, and the radialaxis (i.e., the through axis) of each hole is parallel to the walls ofthe chamber and perpendicular to the faces of the plate. In a morepreferred embodiment, the holes have the shape of right circularcylinders. See, for example, FIG. 1. In another preferred embodiment,they have the shape of a right polygonal prism. The holes can bearranged in any configuration that is suitable to an experiment, e.g.,an array of one or more rows and one or more columns, the array being asquare array of holes, a triangular array of holes or anotherconfiguration of holes. The surface of the plate can be prepared ortreated so that it repels water or other aqueous solutions, thusensuring that small volumes of sample which are deposited in the holeswill remain in the holes without the possibility of leakage or crosscontact with samples in other holes.

The spacing between the holes can be varied to accommodate the densityand pattern of holes on the substrate, so long as mixing betweenadjacent sample chambers does not occur. The substrate can have fromabout 1 to about 10,000 microholes per apparatus. In preferredembodiments there are at least about 600 microholes per apparatus, morepreferably at least about 800 microholes per apparatus, and even morepreferably at least about 1000 microholes per apparatus.

Evaporation may be minimized by providing an evaporation covering sheeton the planar surfaces of the substrate which covers the through-holesand retains vapors within the reaction chamber. Such a sheet may becomprised of any material which does not interfere with the reactioncontained in the chamber. Such a sheet may be hydrophobic in nature andmay by flexible, such as silicone rubber, or may be substantially rigidsuch as a polymeric or glass cover slide, and may comprise an adhesivesubstance. The cover is preferably optically clear.

The top and bottom surfaces of the substrate may contain raised featureswhich form closed curves circumscribing the openings to some or all ofthe sample chambers contained therein. Such features can be used inconjunction with an evaporation retention sheet to improve thereliability of said sheet to prevent loss of vapor. Preferably suchraised features are very narrow such that under a moderate force a veryhigh pressure is maintained at the interface of said feature(s) and theadjacent evaporation sheet. Additionally, a single raised feature maycircumscribe more that one reaction chamber and thus allow communicationbetween all reaction chambers contained therein.

In a preferred embodiment, the apparatus is substantially free ofamplifiable contaminating polynucleotides, particularly for reactions inwhich contaminating amplifiable polynucleotides may interfere with thereaction, e.g. PCR. Preferably, the apparatus has less than 1000amplifiable contaminating polynucleotides per reaction chamber, morepreferably less than 10 amplifiable contaminating polynucleotides perreaction-chamber, even more preferably less than 1 amplifiablecontaminating polynucleotides per reaction chamber. Contaminatingamplifiable polynucleotides may be eliminated from the apparatus by, forexample, .gamma.-irradiation. The presence of contaminating amplifiablepolynucleotides may be detected by, for example by performing a controlPCR reaction with no polynucleotide sample; a control reaction whichyields polynucleotides indicates the presence of contaminatingamplifiable polynucleotides.

Substrates

Numerous materials and methods are available for designing an apparatusfor multiple micro-volume liquid samples. Materials that can be used forthe substrate include, but are not limited to, titanium sheet preferablytreated to render the surface hydrophobic, glass plates with chemicallyetched holes and silanated surfaces, plastics, teflon, synthetics,metals and ceramics. Techniques of electrodeposition manufacturing, andprinted-circuit board manufacturing processes can also be used in thefabrication of the apparatus. In one embodiment, the substrate has ahydrophobic surface and each sample chamber has hydrophilic interiorwalls. In another embodiment the hydrophilic region of the interior ofat least one of the sample chambers extends to the surface of thesubstrate and extends beyond the orifice defined by the sample chambersuch that the area occupied by the extended portion is substantiallycontained on the substrate surface and such that the hydrophilic regionof one sample chamber does not contact the hydrophilic region of anyother sample chamber. Such a hydrophilic region may aid in loadingaqueous reactions into the reaction chamber.

Titanium is bio-inert, hydrophilic, and can be chemically etched toprovide a dense array of holes in any pattern desired. It is also verydurable and hence reusable. Photo-etched titanium substrates are alsouseful for fabrication and are available, for example, from Tech-Etch,Plymouth, Mass.

Glass plates rendered hydrophobic by a surface treatment, for example,by silanation are also suitable; since glass is hydrophilic andsilanation renders its surface hydrophobic. Silicon microfabrication isa suitable method for fabrication of apparatuses comprising extremelywell defined, high density arrays of sample chambers.

Advances in the field of printed-circuit (PC) board manufacturing can beapplied to the fabrication of the apparatus. Current printed circuitboard technology provides both miniaturization and cost efficiency.

An additional process which can be used in the fabrication of anapparatus as disclosed herein is photolithographic electrodeposition.This technique involves slowly depositing metal ions (electroplating)onto a substrate in a photolithographically defined pattern. Thistechnology reliably produces through-holes of 1 .mu.m diameter, and canproduce over 3 million holes per square inch. In one embodiment, thistechnology is used for fabrication of an apparatus comprising very highdensity microhole arrays. Photolithographically fabricated substratesare available, for example, from Metrigraphics, Wilmington, Mass.

Other techniques for fabrication known in the art may be used forconstructing the apparatuses of the invention, for example, lasermicromachining and microfabrication techniques.

Sample Delivery and Recovery

Delivery of samples and reagents to a microhole can be achievedmanually, or through the use of commercially available pipetting robots,such as those available from the Hamilton Company, Reno, Nev. and thePackard Instrument Co., Meriden, Conn. Currently-available pipettingrobots can reliably deposit sample volumes as small as 50 nl, and arobotic positional repeatability of 50 .mu.m is common. Higher accuracyand repeatability can be achieved through special design, such as by thecoupling of piezoelectric and mechanical translation devices (e.g.,Physik Instrumente, Costa Mesa, Calif. For dispensing reagent volumes inthe sub-nanoliter range, piezo-electric pipettors and ink-jet pipettors,for example, can be used.

Thus, reactions can be prepared in the microholes of the apparatus byany means commonly used for dispensing small liquid volumes into smallvessels including, but not limited to: (1) dispensing very small volumesof reagent directly into each hole, either manually or by means of arobotically controlled syringe, (2) immersing the entire apparatus, or apredetermined fraction thereof, directly into a reaction solution,thereby acquiring a volume of reaction solution in each hole that hasbeen immersed, and (3) dispensing, pouring over or flowing over thesurface of the substrate a volume of the reaction mixture. In anotherembodiment, reaction components are affixed within a sample chamber, forexample, by placing a solution within the sample chamber and drying it,such that a solute is affixed to the wall of a sample chamber. Reactionmixtures and/or additional reagents are then added to the chamber,re-solubilizing the affixed reaction component. In preferredembodiments, more than one, more than two, more than three reagents maybe affixed to the wall of a sample chamber. For PCR reactions, forexample, primers, probes, and/or buffer components can be preaffixed tothe sample chambers, allowing for faster preparation times.

After initial addition of reagent, subsequent reagent additions can beconducted. Means for adding additional reagent to a microhole include,but are not limited to, the previously-described methods, as well as:(1) direct dispensing, either manually or by means of, for example, arobotically controlled syringe, (2) direct dispensing into the reactionsolution by a non-contact means such as a piezo-electric dispensingapparatus, (3) deposition of a vaporized solution of reagent, and (4)contact between two or more of the apparatuses, such that material in ahole from one apparatus is transferred wholly or in part to a hole inanother apparatus. In one embodiment, transfer occurs betweencorresponding holes in two or more apparatuses.

An additional exemplary method for sample delivery to sample chambers inan apparatus is shown schematically in FIG. 5. In this embodiment,samples 31 are arranged on a hydrophobic surface 32 in a pattern thatmatches the pattern of holes 33 in an apparatus 34. FIG. 5A. Theapparatus 34 is then brought into proximity with the hydrophobic surface32, such that the samples 31 contact the holes 33 in the apparatus 34.FIG. SB. The apparatus is then withdrawn from proximity with thesurface, the holes 33 now containing samples 31 (FIG. SC).

The process of liquid transfer from one apparatus to another can also beused to make several copies of a single setup plate simultaneously. Forexample, if a group of microhole array plates are stacked, one on top ofanother, and liquid samples are introduced into the top or bottom plate,the samples will wick through the entire stack, thereby generating aseries of plates having identical sample configurations.

Furthermore, contents of a sample chamber, or of an entire apparatus,can undergo dilution, particle size selection, selective retention of amolecule in a sample chamber, desalting, reagent addition, or anotherchemical modification by bringing the apparatus, or a portion thereof,in liquid contact with one or more additional apparatuses, theadditional apparatus(es) prepared in such a way that contact betweenapparatuses will, by chemical 2119/140 diffusion from one sample chamberon one apparatus to the another sample chamber on an adjacent apparatus,achieve the required sample modification. See infra.

After reaction assembly in an apparatus is complete, the apparatus cansubsequently undergo chemical processing and/or incubation in athermally-controlled environment. A concern when dealing with minuteaqueous samples, such as are present in the sample chambers of theapparatus, is evaporation. One way in which this problem can bemitigated is by conducting incubation in a high-humidity environment.For example, various types of water vaporizers are readily available andcan be easily integrated into a laboratory device to provide ahumidified chamber. Other methods of reducing evaporation include, butare not limited to, placing the apparatus in a humidified chamber,maintaining the atmosphere of an open apparatus at saturated vaporpressure, and periodic addition of water to the reactions by means of apiezo-electric or other dispenser in a manner which counteracts theevaporation rate.

Alternatively, the apparatus can be immersed in a hydrophobic mediumsuch as, for example, a bath of an inert liquid that is essentiallynon-miscible with water and which essentially does not react with thesubstrate nor essentially perturb the reaction(s) contained in thesample chamber(s). In many applications an example of a suitablehydrophobic medium is oil, for example, mineral oil or silicone oil.

Reactions contained in the sample chambers of an apparatus can bethermally cycled in such a way as to carry out, for example,amplification reactions such as a polymerase chain reaction (PCR) or DNAsequencing reactions such as, for example, chain-termination sequencingand cycle sequencing. In such thermal cycling reactions, evaporation ofthe sample can be minimized by submerging the sample in a hydrophobicmedium such as, for example, a bath of hydrophobic medium or othersuitable fluid which is maintained at a temperature appropriate for thechemical reaction, by coating the apparatus with a layer of hydrophobicfluid, or by overlaying the samples with a hydrophobic fluid. Forexample, addition of samples to sample chambers can be followed bydirect addition of a film of oil or other hydrophobic fluid to eachsample using a pair of robotic pipets; one filled with a 2119/140 sampleand the other filled with the hydrophobic fluid.

If, for example, an oil bath is used for temperature control,temperature cycling can be achieved by thermally cycling the bath, or byrobotically moving an apparatus from one bath to another bath held at adifferent temperature. Alternatively, thermal cycling of reactionscontained in an array of microholes can be carried out in a humidifiedenvironment, maintained by sealing the array between one or twoevaporation barriers (e.g., silicone sheets or Parafilm.RTM.), andplacing this sandwiched array on a thermoelectric heating block, orbetween two thermoelectric heating blocks. In using an evaporationbarrier such as a silicone sheet, the presence of narrow raised featurescircumscribing each sample chamber or a set of sample chambers to aid inpreventing the loss of vapor by providing a very tight seal around eachhole.

Means for dispensing water to counter evaporation can also be used todispense other reagents or chemicals of interest, mixed in a solutionsuch that evaporation is countered. In this way, chemical assays can becarried out in real time and the evolution of the assay can be directedvia feedback from the assay in progress. To provide but one example,optical data obtained by placing the apparatus on an optical detector,such as a CCD or fiber optics cable, can be input to a computer whichautomatically adjusts reagent dispensers and guides them to dispense aprecise amount of reagent into each sample chamber.

Advantages

The apparatuses disclosed herein, when used, for example, in abiochemical reaction format, provide the advantages of: (1) highdensity, (2) high throughput, (3) ease of handling, (4) performance ofvery low-volume reactions (5) rapid thermal cycling, and (6)advantageous optical access of the samples. In embodiments in which theapparatus is moved from one thermal environment to another, thethin-film nature of the samples ensures very rapid thermal equilibrationtime. In an alternative embodiment in which an apparatus is heldstationary in a thermoelectric (Peltier) device and the temperature ofthe device is changed, apparatuses having a planar symmetry allow twothermoelectric devices to be used, one on each side of the apparatus.This provides a sample ramp speed at least twice that obtained when asingle Peltier device is used, as well as finer control of thetemperature profile within the sample.

Further benefits of the microhole array format of the apparatus includethe ability to transfer liquids from one sample chamber in a firstapparatus to another sample chamber in a second apparatus simply bybringing two apparatuses in close enough proximity to allow the contentsof two sample chambers to touch. Upon physical contact the two sampleswill diffuse together. This technique allows sample mixing, sampledilution and diffusion-based molecular separations e.g., de-salting. Thecontents of a sample chamber in a first apparatus can be transferred, inwhole or in part, to a sample chamber in a second apparatus.

For sample mixing, two apparatuses are brought into contact, as above,such that the liquid contents of one or more pairs of sample chamberscome into liquid contact, wherein one member of each pair of samplechambers is present in a first apparatus and the other member is presentin a second apparatus. Mixing of three or more samples, using three,four, etc. apparatuses is also possible, as will be evident to one ofskill in the art. This is particularly advantageous when being used totransfer nucleic acids, such as DNA and RNA, from one apparatus toanother. Nucleic acids are easily sheared by methods such as pipeting,and this method allows for the transfer of nucleic acids without theneed for pipetting.

For sample dilution, a first apparatus containing one or more samples iscontacted with a second apparatus containing diluent, such that liquidcontact is achieved between one or more sample chambers in the firstapparatus and one or more sample chambers in the second apparatus, andthe apparatuses are allowed to remain in contact for a specified time.In this case, all components in the sample are diluted, with the degreeof dilution depending on the time of contact between the first andsecond apparatuses. If all samples in a first apparatus are to bediluted, the first apparatus need not be contacted with a secondapparatus, but can simply be contacted with a pool of diluent.

Selective retention of a molecule in a sample chamber, dependent ondiffusion-based separation of low molecular weight molecules frommolecules of high molecular weight, is possible using the apparatuses asdisclosed herein. For example, a sample contained in a microhole isdesalted by repeatedly touching the sample, for a short time, to a bath(or to a microhole of another apparatus) containing a solution of verylow salt concentration. Since very small molecules (such as salts)diffuse very rapidly (approximately 60 .mu.m per second), while largermolecules take much longer to diffuse (e.g., a 2 kilobase nucleic acidhas a diffusion rate of approximately 6 .mu.m per second, see, forexample, Smith et al. (1996) Macromolecules 29:1372-1373), an overallreduction in salt concentration of the sample is achieved.

In one embodiment, two apparatuses having identical patterns of samplechambers are contacted so as to bring corresponding sample chambers intocontact. Two (or more) sample chambers are “corresponding” if they arelocated in the same position on different apparatuses (ie., if eachapparatus comprises an array of microholes, corresponding microholesoccupy the same location in the array). In additional embodiments forselective, diffusion-based molecular retention, desalting, dilutionand/or reagent addition, contact between corresponding sample chambersis not required. For example, a single sample-chamber in a firstapparatus can be contacted with multiple, different sample chambers of asecond apparatus. In a separate embodiment, subsets of chambers in afirst apparatus are contacted with sets of chambers in a plurality ofadditional apparatuses.

Another advantage of the apparatus format of the invention is theability to minimize shear when loading a microhole array with a nucleicacid. Nucleic acids, such as DNA and RNA, are easily sheared by transfermethods such as pipetting. The apparatus of the invention may be loadedwith a solution comprising a nucleic acid simply by contacting theapparatus with a liquid solution, for example, contacting the apparatuswith a tray containing the solution of interest (e.g., “dip loading”).

In some embodiments, formation of a thin film by a sample, when it iscontained in a sample chamber of the apparatus, results in.a ratio ofsurface area to volume that facilitates optical analysis of the sample,either continuously during the reaction period or at one or severalpredetermined time points. Because photons pass through a minimum fluidvolume in the thin film, more efficient detection of light absorptionand emission (e.g., fluorescent, chemiluminescent) by a sample ispossible. Furthermore, because of these favorable optical properties,progress of multiple reactions can be monitored in real time (i.e.,during the course of the reaction). For example, in reactions thatgenerate an optical signal, e.g. a colored, fluorescent, or luminescentproduct, reaction progress can be monitored in real time for instanceusing multiple optical detectors aligned with the sample chambers, fiberoptics, a detector that scans across the apparatus, or a whole-apparatusimager. Compared to analysis of micro-volume reactions in capillaries,the apparatuses disclosed herein allow improved optical analysis that isnot prone to either refractive effects from capillary walls or opticalcross-talk between neighboring capillaries.

Applications

Apparatuses as disclosed herein can be used for holding and arraying anytype of liquid micro-volume sample. They can also be used for performingany type of biochemical or molecular biological reaction known to one ofskill in the art including, but not limited to, nucleotide sequencing(e.g., chain-termination sequencing, cycle sequencing), amplificationreactions (e.g., polymerase chain reactions), transcription, reversetranscription, restriction enzyme digestion, ligation, primer extension,other enzymatic reactions and biological interactions (such as, forexample, avidin-biotin, streptavidin-biotin, antibody-antigen andligand-receptor interactions). In general, any type of enzyme-mediatedreaction can be performed in the apparatus. In addition, multiplemicro-volume hybridization reactions can be conducted in the apparatus.In one embodiment, an apparatus is used for very high throughputanalysis of chemical samples; for example, in combinatorial chemistry.Several examplary applications are disclosed below, including those inthe Examples, and additional applications are known to those of skill inthe art. Use of the apparatuses disclosed herein will be especiallyuseful in the field of genetic analysis, for techniques such aspolymorphism detection (see infra).

Amplification Reactions

Very high throughput of small volume amplification reactions, such aspolymerase chain reactions, ligase chain reactions, rolling circleamplification, and “Taqman.RTM.” hydrolyzable probe assays is obtainedusing, for example, an apparatus containing an array of microholes. Theability to perform a large number of individual reactions, each in avery small volume, obviates the need for multiplex PCR (in which severaldifferent genomic loci are amplified in a single reaction) and avoidsthe technical difficulties inherent in that strategy. Alternatively,low-number multiplex reactions can be carried out in a microhole format,compounding the benefits of this technology.

In many applications of PCR, recovery of an amplification product isdesirable. Problems with recovery of amplification products usingmethods of the prior art are related to the elevated temperatures usedfor most amplification reactions, necessitating the use of an oiloverlay to prevent evaporation of the reaction mixture. In these cases,the presence of oil can interfere with recovery of the amplificationproduct(s), for example, making it difficult to aspirate a microvolumesample. Methods which do not require the use of oil (e.g., conductingamplification reactions in capillaries) still present problems withfluid manipulation.

This problem can be addressed by use of apparatuses as disclosed herein.For example, a porous hydrophobic membrane that is preferablyessentially non-reactive (such as a teflon membrane filter) can be usedin conjunction with an apparatus. In this embodiment, an apparatuscontaining a plurality of reactions is immersed in an oil bath forconducting a high-temperature reaction, removed from the bath, andtouched to the porous hydrophobic membrane. The hydrophobic medium willreadily wet the hydrophobic membrane and flow into it, whereas anaqueous reaction solution will be repelled. Subsequent removal of theapparatus from the hydrophobic membrane leads to the formation ofdiscrete drops of aqueous solution resting thereon. These aqueous dropscan be readily accessed. Alternatively, direct pipetting of an aqueousreaction solution from beneath an hydrophobic substance layer onhydrophobic surface is possible with accurate and reliable robotics.

Moreover, for certain applications, the presence of a hydrophobicsubstance is not a hindrance. For example, when an apparatus is used forloading reaction products into an acrylamide gel (see infra), theapparatus (optionally having been used as a reaction sheet) can beplaced onto the top of a gel and overlayed with upper reservoir buffer.After addition of buffer, the hydrophobic substance separates from thebuffer layer, thereby separating from the gel samples.

Molecular Haplotyping

Knowing the haplotype, or the “phase” of the genotypes of an individualis far more informative than simply knowing the genotypes alone.Standard methods of haplotyping involve a statistical analysis of thegenotype distributions, often relying on assumptions regarding therecombination rate of the genetic region and the size of the recombinedregion. The apparatus and methods described herein may be advantageouslyused to experimentally identify the haplotype.

Following the digital-PCR application outlined by Vogelstein and Kinzler(PNAS, vol.96, p. 9236-9241), the microhole apparatuses described hereinmay be used to provide high confidence haplotype information byperforming hundreds or thousands of PCR reactions simultaneously on aDNA template.

The technique requires individual amplifications of single molecules ofDNA. Such single molecule amplification can only be achieved on theaverage by applying a terminally diluted sample of template DNA to anarray of microholes. On average half of the holes will contain exactlyzero copies of the DNA region of interest, somewhat less than half willhave exactly one copy, and fewer still will have two or more copies. Thedistribution of number of holes with a given number of templates shouldfollow a Poisson statistical distribution. DNA samples may be prepared,for example, by using the Stratagene DNA Extraction kit (Stratagene, LaJolla, Calif.) according to manufacturer's instructions, and thendiluted to a concentration of one-half genome equivalent per reaction.

The tests may be performed, for example, by using fluorescent duallabelled probes and the 5-prime exonuclease (TaqMan) assay. In thisassay a probe is added to the PCR brew which is designed to hybridize toa sequence of interest within the PCR amplicon. The probe is synthesizedwith two fluorescent molecules-an emitter and a quencher. These twomolecules are each attached to individual bases on the probe and arespaced typically 5-7 bases apart so that the quencher molecule prohibitsthe emitter from fluorescing. As a single unit the dual-labelled probeis non-fluorescing. During the PCR process a probe molecule willhybridize to the template molecule which is being polymerized, and asthe Taq polymerase synthesizes the complement to the template, the5-prime to 3-prime exonuclease activity of the Taq polymerase willdegrade and displace the hybridized primer. This degradation separatesthe emitter from the quencher molecule and thus allows the emittermolecule to fluoresce. This emission can be read by a standardfluorescent plate reader and the intensity of the fluorescence isgenerally quantitatively related to the number of initial templatemolecules. Probe molecules which do not stringently hybridize are notdegraded and will not fluoresce.

Thus, the final distribution of the sequence(s) of interest may beobtained by counting the number of single fluorescent unit intensitymicroholes versus zero intensity microholes (where a unit of intensityis derived by comparing the brightness of each hole; the holes whichstarted out with two templates should have approximately twice thebrightness of most of the remaining fluorescent microholes, which shouldhave started with only a single template molecule).

An apparatus (or “chip”) of the invention may be loaded with theappropriate PCR reactants, for example, by dip-loading a solutioncontaining the PCR reactants, or by pipetting (preferably with a largebore pipet) the solution(s) into the microholes. The apparatus may bepreloaded with one or more reactants. The apparatus is preferably sealedwith, for example, a single sheet of clear adhesive film (such as parcelpackaging tape) folded around one edge and sealed at the opposite edgewith a face-to-face adhesive seal.

The apparatus is thermal cycled in a thermal cycler which may bemodified by milling or sawing a narrow, deep groove across its face sothat the thin microhole chip may be inserted into the groove and thechip reliably thermal cycled. Preferably, a thin heat-conductivesilicone pad (Stockwell Rubber Company, Philadelphia, Pa.) is used toprovide thermal contact as well as pressure to the surfaces of themicrohole chip. The added pressure prevents evaporation of the samples.

Following thermal cycling, the chip is scanned using a scanner orfluorescent microscope to determine the levels of fluorescence, in orderto determine the final distribution of the sequence(s) of interest.

Very High Throughput PCR

High throughput PCR is readily achieved using, for example, microholearrays by dispensing template, reagent and primer pairs to eachmicrohole. Typically, two of these steps are combined: for instance thetemplate and reagent (enzyme, buffer, etc.) are combined in a master mixand the master mix is loaded simultaneously into all microholes bydipping an apparatus into a solution of master mix or by spraying asolution of master mix over an apparatus such that the solution entersthe sample chambers. A significant increase in throughput is achieved bypre-affixing oligonucleotide primers and probes to each chamber, or bypre-synthesis of all primer pairs in a microhole array. In this case,reactions can then be assembled simply by immersing a pre-synthesizedmicrohole array plate into a bath of master mix.

Technologies for pre-synthesis of primers on the apparatus include:standard phosphoramidite and photo-phosphoramidite chemistries. Standardphosphoramidite chemistry is used for most oligonucleotide synthesisoperations when it is necessary to recover the oligonucleotide insolution for later use. See, for example, U.S. Pat. Nos. 4,415,732;4,458,066; 4,500,707; 4,973,679; and 5,153,319. Photo-phosphoramiditechemistry, for synthesizing oligonucleotides on a solid substrate forlater use on that substrate, has been disclosed, for example, in U.S.Pat. Nos. 5,445,934; 5,510,270; and 5,744,101; and PCT publication WO99/19510.

Using photo-phosphoramidite chemistry, substrates containing up to10,000 discrete oligonucleotides can be obtained. In one embodiment, anoutput array is controlled by an array of micro-mirrors and opticalelements and is highly flexible. Singh-Gasson et al. (1999) NatureBiotechnology 17:974-978. Thus, this technique is suitable for formingarrays for use in highly parallel processing and can be adapted forsynthesizing oligonucleotides in, for example, an array of microholes.

Cycle Sequencing

Small volume cycle sequencing reactions can be performed in a highthroughput setting, in a fashion similar to the PCR application, supra.An additional benefit of the technology disclosed herein is the abilityto perform sequencing reactions directly on the comb or plate that willbe used for loading the reaction product onto the sequencing apparatus.For instance, for using standard slab gel electrophoresis sequencingapparatus, reactions are performed in a 1-dimensional array of holeswhich has been pre-formed on a gel loading comb. See, for example, Erfleet al. (1997) Nucleic Acids Res. 25(11):2229-2230. Sequencing reactionsare assembled in the array of holes on the comb and, after completion ofthermal cycling, the reaction product is directly loaded onto a gel forelectrophoresis. FIG. 6 depicts several embodiments of this type ofarray. FIG. 6A shows an apparatus 40 in which the sample chambers 41 arelocated close to one edge 42 of the apparatus. FIG. 6B shows a portionof an apparatus 50 in which the sample chambers 51 communicate with theexterior of the apparatus. FIG. 6C shows a portion of an apparatus 60 inwhich the sample chambers 61 communicate with the exterior of theapparatus via channels 62. Additional configurations of the apparatussuitable for gel loading will be apparent to those of skill in the art.

Endonuclease Reactions

Standard room temperature or elevated temperature restrictionendonuclease digestions can be performed using the disclosedapparatuses. An endonuclease reagent master-mix is loaded into eachsample chamber, either by automated pipetting means or by immersing, forexample, a microhole array into a bath of reagent. Subsequently,individual samples of, for example, nucleic acid and/or restrictionendonuclease, can be loaded into each well using an automated pipettoror other means. After the reactions are assembled the apparatus isincubated at a temperature appropriate for the assay. If the durationand temperature of the incubation is such that evaporation of thesamples may be a problem, the incubation can take place in a humidifiedchamber or under a hydrophobic substance to counter or mitigate theeffects of evaporation. See supra.

Biological Interactions

The strong biochemical interaction between biotin and streptavidin (oravidin) has made these molecules useful for binding assays. For example,incorporation of a biotin-labeled nucleotide into a polynucleotide, andsubsequent capture of the polynucleotide with streptavidin, is a commonmethod for isolating a specific polynucleotide sequence. Abiotin-streptavidin capture is easily performed using the disclosedapparatus by simply touching an apparatus, optionally containingbiotin-labeled samples, to a plate or substrate which has streptavidinbound to its surface. The plate can be a membrane, microscope slide,cover slip, or another microhole array with streptavidin bound to theinner wall of the sample chambers.

Other pairs of interacting molecules can also be used in a similarfashion. Examples include, but are not limited to, antigen-antibody,hapten-antibody, sugar-lectin, and ligand-receptor.

Oligonucleotide Synthesis

In another embodiment for performing chemical reactions, an apparatussuch as a microhole array is used as a miniature oligonucleotidesynthesizer, utilizing standard phosphoramidite chemistry and electricaladdressing. It is a straightforward extension of microfabricationtechnologies as used in integrated circuit production to design amicrohole array in which each hole can be individually charged oruncharged. See, for example, U.S. Pat. Nos. 5,605,662; 5,632,957; and5,929,208 for related techniques used in the construction of amicroarray. Using such techniques, a different oligonucleotide can besynthesized at each hole in a multi-step process. At each step, thearray is exposed to a nucleotide monomer, and the sites on the arraycontaining oligomers to which that monomer is to be added areelectrically addressed so as to direct the monomer to those sites.

Such a device would have the multiple advantages of producingoligonucleotides in a predetermined location as well as producingsmaller amounts of oligonucleotide required for a particular reaction,leading to increased economy and efficiency.

Genetic Analysis

An important result of the efforts to determine human (and other) genomesequences is the availability of a vast pool of genetic data (in theform of DNA sequence) which can be subjected to a multitude of geneticanalyses. The results of the various genetic analyses can be applied todiagnostic, pharmacogenomic and therapeutic applications, to name but afew. One particularly valuable form of genetic information-that isavailable through the analysis of DNA sequence is genetic polymorphism.Polymorphism can be due to insertion, deletion, translocation,transposition and/or tandem repetition of particular portions of asequence, or to single- or multiple-nucleotide changes at particularpositions within a sequence.

Many methods known in the art can be used to determine the presence of agenetic polymorphism. For example, insertions and deletions, as well assome types of transposition and translocation, can be detected byrestriction fragment length polymorphisms (RFLPs).

Another method for determining the presence of a polymorphism is byanalysis of tandem repeat lengths in minisatellite DNA. This techniqueinvolves restriction enzyme digestion and blot hybridization, and/orSTRP analysis, which involves PCR, gel electrophoresis or primerextension and mass spectrometry. See, for example, Birren et al. (eds.)“Genome Analysis: A Laboratory Manual” Cold Spring Harbor LaboratoryPress, 1999, esp. Volume 4.

Polymorphisms resulting from a single nucleotide change (“SNP”) may ormay not result in a change in the size of a restriction fragment. Amultitude of additional techniques, known to those of skill in the art,are available for the detection of SNPs. These include, but are notlimited to, denaturing gradient gel electrophoresis, single-strandconformation polymorphism analysis, heteroduplex analysis, temperaturegradient gel electrophoresis, cleavase-fragment length polymorphism,denaturing HPLC, chemical cleavage of mismatch, carbodiimidemodification, enzymatic cleavage of mismatch,uracil-N-glycosylase-mediated T scan, direct nucleotide sequencing, DNAchip resequencing, allele-specific primer extension, oligonucleotideligation assay, randomly amplified polymorphic DNA analysis(“RAPD”),fluorescence energy transfer dye terminator incorporation assay (“FRETTDI”), dye-labeled oligonucleotide ligation assay (“DOL”), Taqman® withallele-specific oligonucleotides, randomly amplified polymorphic DNAs,and analysis by the Invader® (Third Wave Technologies) technique.Additional methods of polymorphism analysis are known to those of skillin the art. See, for example, Birren et al., supra.

In an embodiment-of the invention, genetic polymorphism analysis can becarried out as described supra, in the apparatuses disclosed herein,which will thus be useful in these types of genetic analysis.

A method for SNP determination is by single base primer extension. Inthis technique, a primer is annealed to a polynucleotide that is to betested for the presence of a SNP. The sequence of the primer is chosensuch that the 3′-terminal nucleotide of the primer is adjacent to thesite that is to be tested for the presence of the SNP. Thistemplate-primer complex is used for the preparation of four separateprimer extension reactions, each containing only a single nucleotide.The reaction(s) in which extension occurs provides the sequence of thesite being tested for the presence of the SNP. See, for example, U.S.Pat. No. 6,004,744.

Although extension can be assayed by DNA sequencing techniques,alternative assays are known in the art. One alternative assay forextension measures increases in molecular weight by mass spectroscopy,for example, matrix assisted laser desorption ionization time-of-flight(MALDI-TOF) spectrometry. Primers that have been extended by a singlenucleotide, having a higher molecular weight, will be distinguished fromunextended primers when analyzed by MALDI-TOF or other forms of massspectrometry.

The apparatuses disclosed herein are useful in MALDI-TOF, and othertypes of mass spectrometric analyses, because transfer of extensionproducts to a mass spectrometry preparation platform can be achieved bytouching rather than pipetting. Consequently, multiple samples can betransferred simultaneously. Rapid sample preparation for multiple massspectrophotometric analyses is accomplished by dehydration concentrationof a sample on a hydrophilic region of an apparatus. The hydrophilicregion can be located, for example, on the wall of a sample chamber.

The discovery of SNPs is efficiently accomplished by direct nucleotidesequence determination. The apparatuses disclosed herein provide anideal route to SNP discovery by facilitating high-throughput nucleotidesequence determination.

Sandwiched Reactions

Chemical and biochemical reactions can be assembled by placingapparatuses containing individually pre-filled sample chambers adjacentto each other in such a way that corresponding samples in differentapparatuses attain physical contact and the contents of thecorresponding samples mix spontaneously. In one embodiment, the samplechambers in an apparatus are pre-loaded and allowed to dry so that thecontents of the chambers (e.g., oligonucleotide primers) are in a stateof dehydration. When such a dehydrated apparatus is brought into contactwith an apparatus whose sample chambers are loaded with an aqueousreaction component, the dehydrated component(s) will be re-hydrated.This method can be practiced with several apparatuses at a time,allowing complex reactions to be assembled. A further advantage is thatthis method minimizes effects of evaporation that occur during reactionassembly, because all sample chambers are re-hydrated simultaneouslywhen a stack of, for example, microhole arrays is assembled. Reactionscan be conducted in a plurality of apparatuses arranged in a stacked orsandwiched configuration.

Addition of Reagents During Chemical Processing

Reagents can be added to reactions in progress in an apparatus, duringincubation, by means of a piezo-electric dispenser or other commonapparatus. In this way, the effects of evaporation can be countered bythe addition of water; alternatively, chemicals and/or enzymes can beadded to a reaction in progress. Using this method, reactions can beoptimized as they proceed (i.e., in real-time).

Library Display and Assay

The apparatus of the invention can be used for creating and displayinglibraries, for example, libraries of cells. For example, the samplechambers can comprise an adherent surface, suitable for cell growth,such as plastic, polystyrene and optionally polylysine. A differentcell, cell strain or cell type can be applied to each sample chamber andthe substrate placed under conditions suitable for cell growth; forexample, the substrate is immersed in culture medium in a CO.sub.2incubator at 37.degree. C. After a period of cell growth, the substrateis removed from the growth conditions and subjected to conditions thatresult in cell lysis and fixation of cellular macromolecules within oradjacent to the sample chambers. The substrate can then be subjected to,for example, restriction enzyme digestion, hybridization and/oramplification analysis to determine the presence of a particular targetmacromolecule in a particular cell, for example.

Real-time Analysis of Reactions

Optical monitoring of the reactions contained in the sample chambers ofthe apparatus can be achieved in several ways. For example, an array ofmicroholes containing a plurality of completed reactions can be placedin direct contact with a CCD array or fiber optics bundle, or can beloaded into an optical reading apparatus. For light-emitting readouts,such as fluorescence or chemiluminescence, a benefit of the microholearray is that there may be no plastic or other material to obscure ordiffuse the emitted light and potentially generate autofluorescence. Forthermal cycling reactions contained in a hydrophobic medium such as abath of a hydrophobic substance, it is possible to monitor the opticalactivity of the reaction continuously. The hydrophobic substance itselfcan optically couple the reaction to a fiber optic bundle immersed inthe hydrophobic substance which directs emitted light to a CCD array forquantitative detection. Such a device allows very high parallelprocessing of real-time assays such as PCR and Taqman®. Using fiberoptic bundles capable of carrying hundreds of thousands to millions ofindividual fibers, it is possible to monitor millions of amplificationreactions simultaneously and in real-time on a single apparatus such asa microhole plate.

High Throughput Sequencing

In addition to providing significant advantages in cycle sequencing, asdescribed above, the use of the disclosed apparatuses, such as microholearrays, for chemical reactions provides advantages in standardnucleotide sequencing operations as well. For example, it is possible todesign a microhole sheet that is thin enough to fit between the glassplates of a standard polyacrylamide slab gel. The sheet has microholesarranged in a straight line array with each microhole containing asub-microliter volume of a chain termination sequencing reaction. See,for example, FIG. 6A. The sheet is placed into a constant temperature orthermal cycling apparatus for processing of the sequencing reaction, andis then transferred to the top of a polyacrylamide gel or otherelectrophoresis medium, with one edge of the sheet contacting theelectrophoresis medium. In one embodiment, the sheet contains partialholes along its perimeter into which reaction mixtures are dispensed andreactions are conducted, and from which reaction mixtures are applieddirectly to a gel by the method described above. A partial hole is onewhich is not completely enclosed by the substrate, such that up to about180.degree. of the diameter of the hole is not enclosed by thesubstrate, i.e., up to 180.degree. of the hole diameter is open to theexterior of the substrate. See, for example, FIG. 6B. Alternatively, ahole can communicate with the exterior of the substrate through a thinchannel in the substrate. See, for example, FIG. 6C.

For sequencing techniques in which the termination productscorresponding to a particular one of the four nucleotides are labeledwith a chromophore or fluorophore specific to that nucleotide, it ispossible to combine the four base-specific reactions for analysis on asingle gel lane. In this case, four arrays, each containing a differentone of the four sequencing reactions in a corresponding hole, arestacked, and the stack is placed in contact with a sequencing gel suchthat the samples enter the gel upon provision of an electric current.Alternatively, the four plates are stacked so that the four sequencingreactions mix and the mixture equilibrates throughout the stack. Thenone of the plates is removed from the stack and placed in contact withthe gel.

Use of an apparatus such as a microhole sheet for gel loading has manyadvantages. The very small reaction sizes available with the sheetsresults in reduced reagent usage and consequent cost savings.Additionally, a sheet can be pre-loaded with hundreds of sequencingreactions, rather than the current limit of 96 samples per gel, therebysignificantly expanding the capacity and throughput of current gel-basedsequencing techniques and exceeding those of capillary sequencinginstruments.

Additional Electrophoretic Applications

In another embodiment of the use of the disclosed apparatuses forelectrophoresis, a microhole array containing a plurality of samples isplaced such that one face of the array is in contact with anelectrophoresis medium. In this way, several rows of samples can besimultaneously transferred to the electrophoretic medium to provide athree-dimensional electrophoretic analysis. Detection of samples duringand/or after electrophoresis is accomplished, for example, byfluorescence. In one embodiment, a molecule which becomes fluorescentupon DNA binding (such as ethidium bromide, acridine orange or SYBRgreen, for example) is present in the electrophoretic medium. In anotherembodiment, the sample being subjected to analysis is labeled with afluorescent molecule. In another embodiment, samples are radioactivelylabeled and detected with a radiation scanning device. In addition, theycan be detected by silverstaining, Coomassie blue staining, and bybinding of other proteins (e.g., antibodies).

In another embodiment, a stack of one or more gel- or liquid-filledmicrohole arrays can be formed to simulate an array of capillaries. Anarray containing a plurality of reactions can be placed upon the stack,and the reactions electrophoresed out of the array and into the stack.Subsequent to electrophoresis, the stack can be disassembled and thepresence of a molecule in a particular array can be correlated with theposition (level) of that array in the stack. The thickness of each arrayin the stack need not be uniform, although, in one embodiment, the stackcomprises a plurality of arrays of uniform thickness. The holes in thestack can be filled with agarose, acrylamide, or any other mediumsuitable for electrophoresis. A similar stacking format can be used, forexample, to conduct unit gravity sedimentation through a liquid.

Kits

Included within the embodiments of the invention are kits comprising theapparatus for containing multiple micro-volume liquid samples asdescribed Supra. The kits may also comprise a component of a reaction tobe carried out in the apparatus; the component may be either a reactantor a reagent. In some embodiments, the reactant and/or reagent may becontained within one or more microholes of the apparatus. The kit mayalso contain in addition to the microhole apparatus, one or morehydrophobic substances to be used with the apparatus. The hydrophobicsubstances may be a hydrophobic fluid and/or a solid hydrophobic cover(e.g., a teflon porous membrane and/or evaporation seals (e.g. opticallyclear silicone sheets). Additional contents of the kit may be a chamberfor maintaining the appropriate environmental conditions, e.g., humidityand/or temperature, for the reaction(s) that are to be carried out usingthe microhole apparatus, and an apparatus(s) for loading the samplesinto the sample chambers. When the contents of the kit include a fluidsubstance, the fluid will be packaged in an appropriate container.Desirably, the kit will also contain instructions for use of themicrohole apparatus.

The invention is further illustrated by the following nonlimitingexamples.

EXAMPLES Example 1

PCR-Mediated Analysis of CAG Repeat Length in the Human hSK Gene

A 10 ml aliquot of 2× PCR mastermix is prepared from commerciallyavailable components (e.g. GeneAmp® PCR Reagent Kit with AmpliTaq® DNAPolymerase; PE Biosystems, Foster City, Calif.) and customoligonucleotide primers.

The master mix contains each of the following components at 1.6-timesthe desired final concentration:

Deoxynucleotides (dATP, dCTP, dGTP, TTP (or UTP)) A forward primer:FrwCAG2: GGA CCC TCG CTG CAG CCT CA A reverse primer: RewCAG2: GCA AGTGGT CAT TGA GAT TGA GCT GCC T

A thermostable DNA polymerase (e.g. AmpliTaq® DNA Polymerase)

A buffer

MgCl₂

Using a Hamilton 4000 robot, 1.7 μl of mastermix is dispensed into eachof 576 microholes (24 rows and 24 columns) in a solid substrate (themicrohole apparatus). The holes occur in the shape of a right circularcylinder of 1.2 mm diameter and 1.6 mm height. The microhole apparatusis suspended such that its lower face is shallowly immersed in mineraloil at a depth such that no mineral oil is forced into the microholes.Immediately following dispensing of the master mix, 1 μL of mineral oilis dispensed on top of each microhole.

Alternatively the apparatus is touched or immersed in a reservoir ofmaster mix so that each microhole is filled with mastermix (internalvolume of 1.7 μl). The apparatus is then shallowly immersed in mineraloil so that it is submerged to a depth of <1 mm.

Template DNA is prepared from blood or other tissue(s) from humanpatients of interest e.g., using QIAamp 96 DNA Blood BioRobot Kit(QIAGEN Inc., Valencia, Calif.). Using a Hamilton 4000 robot, 1 μl ofDNA is removed from its position in a 96-well dish and is pipettedthrough the mineral oil into each microhole. Each microhole contains adistinct template DNA sample (derived from an individual patient) buteach patient sample can be assayed multiple times in differentmicroholes.

Following dispensing of the template DNA, the apparatus is immersed in asmall volume (1-5 ml) of mineral oil. The mineral oil is thermallycycled as follows:

94° C. for 40 seconds, 4 cycles of (94° C. for 10 seconds, 70° C. for 50seconds), cycles of (94° C. for 10 seconds, 68° C. for 50 seconds).After thermocycling, the substrate is removed from the oil bath andloading dye (which can contain bromphenol blue, xylene cyanole FF and/orFicoll (commercially available from e.g., BiolOl, Inc., Carlsbad,Calif.)) is dispensed into each microhole either using a Hamilton 4000robot or by hand using an adjustable distance multichannel pipettor suchas the Matrix Impact EXP. The material in the microholes is thenaspirated and samples are dispensed into the wells of an 8%polyacrylamide gel and are electrophoresed to resolve differently sizedproducts.

Example 2

Expression Analysis Template MRNA (or total RNA) is prepared from thetissue(s) of human patients of interest e.g. using an RNeasy 96 BioRobotKit (QIAGEN Inc., Valencia, Calif.). The RNA is combined with a mixcontaining reagents for reverse transcription and PCR amplification(e.g., GeneAmp® Gold RNA PCR Reagent Kit, PE Biosystems, Foster City,Calif.), excluding sequence-specific oligonucleotides and probes, suchthat the reagents are present at 1.6 times the desired finalconcentration. 1.7 μl of template/reagent mix is dispensed into each of576 microholes in a microhole apparatus (the apparatus, as describedabove). Immediately following dispensing of the template, 1 μl ofmineral oil is dispensed on top of each microhole.

Alternatively, the apparatus is touched or immersed in a reservoir oftemplate RNA/reagent mix so that each microhole is filled withRNA/reagent mix (volume 1.7 μl). The substrate is then shallowlyimmersed in mineral oil so that it is submerged to a depth of <1 mm.

PCR primer/probe combinations corresponding to genes of interest aredesigned using e.g. Primer Express software (PE Biosystems, Foster City,Calif.). The fluorogenic probe for each sequence consists of anoligonucleotide with both reporter and quencher dye attached. Each probeanneals specifically between the forward and reverse amplificationprimers. When the probe is cleaved by the 5′ nuclease activity of a DNApolymerase (e.g. AmpliTaq® DNA Polymerase; PE Biosystems, Foster City,Calif.), the reporter dye is separated from the quencher dye and asequence-specific fluorescent signal is generated. The fluorescenceintensity of the dye is proportional to the amount of starting materialpresent in a patient sample.

Cognate PCR primers and probes corresponding to each gene of interestare mixed together at 2.7-fold the desired final concentration. 1 μl ofeach of the individual primer/probe mixes are dispensed into themicroholes containing the RNA template/reagent mix using a Hamilton 4000robot. Each microhole contains a distinct primer/probe combination(corresponding to an individual gene) but each gene can be assayedmultiple times using different microholes.

Alternatively, primers and probes can be prepared at 2-fold the desiredfinal concentration and can be dispensed into empty microholes of aseparate apparatus. The primer and probe mixes are introduced into theRNA template/reagent mix by touching the two apparatuses together toallow mixing of the reagents.

Alternatively the desired amount of primers and probes can be dispensedinto empty microholes, following which the apparatus is dessicated,allowing the primers/probe mix to dry onto the wall of each microhole.The entire apparatus is then touched or immersed in a reservoir oftemplate RNA/reagent mix, so that each microhole is filled withRNA/reagent mix (volume 1.7 μl) as described above. The RNA/reagent mixrehydrates the dessicated primer/probe mix.

Following the introduction of template DNA, the substrate is immersed ina small volume (1-5 ml) of mineral oil. The mineral oil is thermallycycled as follows:

95° C. for 10 minutes, 20-40 cycles of (95° C. for 15 seconds, 60° C.for 60 seconds). After thermocycling, the apparatus is removed from theoil bath, covered on both sides with microscope slide coverslips andscanned on a confocal microscope. The substrate can be returned to theoil bath for additional cycling after scanning, if desired.Alternatively a device can be used to scan the reaction during eachcycle of PCR while cycling is occurring.

Example 3

Microhole PCR with Pre-Affixed Oligonucleotide Primers and In-SituFluorescent Detection

PCR amplification of Lambda phage was performed in a microhole arraywith hole diameters of 1.0 mm and a titanium plate thickness of 1.1 mm.The titanium plate 1 was wrapped at the ends with aluminum tape 71 (FIG.7) to ensure thermal contact of the plate to the heat block of amodified Perkin-Elmer 480 (PE480) thermal cycler while preventingphysical contact between the heat block and the reactions contained inthe holes. The PE480 thermal cycler was modified by cutting a channelacross the width of the heater block to allow insertion and thermalcycling of microhole array plates. The channel was 2.4 mm wide, 18.8 mmdeep and cut across the entire face of the heat block, a distance of 90mm. The open ends of the channel were filled with heat conductivesilicone caulk (Ultra Copper, Loctite Corporation, Rocky Hill, Conn.)and the cured assembly was filled with laboratory grade mineral oil(Sigma Chemical #M-5904).

Primers were designed to amplify a 632 bp region of the Lambda DNA,forward primer=tggtatgaccggcatcct, reverse primer=tcggcgtgtcatatttcact.Initial loading of primer pairs onto the dried titanium microhole platewas 0.5 μl of 10 μmolar dilutions, giving 5 picomoles of forward andreverse primer in each well. The loaded reaction assembly was placedonto a 95° C. hot plate, the aluminum tape at the ends of the titaniumplate providing a thermal path for increased evaporation rate whilecreating a standoff so that the loaded microholes would not makephysical contact to the surface of the hot plate. After several minutes,the dried plate was removed.

A reaction mixture of 1 μl picogreen (Molecular Probes, Eugene, Oreg.),1 ng Lambda Control DNA (AB Gene, Surrey, UK), 3 .mu.l TE(pH=7.5), and 5il 2× master mix was prepared. The 2× master mix consisted of 10 μl 10×buffer concentration, 10 μl MgCl₂ (for a final concentration of 2.5 mM),5 units RedTaq (Sigma Chemical #D-2812), 12 μl dNTPs (1.25 micromolar ofeach A,C,G,T). 0.5 μl of this reaction mixture was added to each of theholes in the microhole array, and thermal cycling was performed.

Thermal cycling parameters for the PE480 were as follows: 3 minutes at95° C., 30 cycles of 95° C. for 30 secs, 55° C. for 30 secs, 73° C. for60 sec, and 10 minutes at 72° C. After thermal cycling the microholearray was removed from the thermal cycler and placed on a confocal laserscanner/imager without removing the oil from the titanium substrate. Thein-house imager/scanner uses a pair of coupled screw-drive robotictranslation stages to scan the microhole array substrate in the x-andy-directions. An Argon-ion laser is focused through an objective lensonto the substrate, the excited emission returns through the objectivelens and dichroic mirrors direct light of 530 nm(±20 nm) wavelength and570 nm(±20 nm) wavelength to two separate photomultiplier tubes (PMTs).The signal of the PMTs is collected and displayed on the attachedcomputer.

The argon-ion laser excited the picogreen (which strongly fluoresces inthe presence of double stranded DNA, but fluoresces only weakly in thepresence of RNA and single stranded DNA) at 488 nm. A fluorescent signalfrom these reactions which is higher than pre-calibrated backgroundsignals indicates successful rehydration of the primers andamplification of the Lambda template.

Example 4

Molecular Haplotyping

Following the digital-PCR application outlined by Vogelstein and Kinzier(PNAS, vol.96, p. 9236-9241), the microhole apparatuses described hereinmay be used to provide high confidence haplotype information. In thisexample we investigate the coincidence of a rare single base changewhich occurs in intron 34 of the ATM gene (IVS34-7 T>C) with a morecommon polymorphism which occurs in intron 48 of the same gene, aninsertion of the three base sequence ATT (IVS48-69 insATT) (Thorstenson,Y R et al. American Journal of Human Genetics, vol.69:396-412,2001 ).

Samples of human DNA are prepared using the Stratagene DNA Extractionkit (CAT# 200600, Stratagene, La Jolla, Calif.) according tomanufacturer's instructions. The purified product is diluted toapproximately 1.5 pg/μl to obtain a concentration of one-half genomeequivalent DNA per 1 μl reaction.

A 256 hole microhole chip is used for this example. A 0.5 mm thicktitanium plate with hole diameters of 0.75 mm will hold liquid samplesof slightly less than 0.9 μl. 250 μl of a PCR brew are prepared asfollows: 67 mM Tris (pH 8.8), 16.6 mM NH4SO₄, 10 mM β-mercaptoethanol, 1mM each A, C, G, T dNTPs, 6% (vol/vol) DMSO, 1 μM each forward andreverse primer, 1 μM each sequence probe (primers and probes describedbelow), 12.5 U Platinum Taq (Life Technologies, Grand Island, N.Y.), and125 genome equivalents of template DNA. Using a large bore 1-ml pipette,the reaction mixture is deposited on top of the horizontally heldmicrohole array. The chip is re-oriented to be vertical, and theoverflow liquid is carefully re-aspirated from each side of the chipusing a 200-μl pipette. The top and bottom of the chip are sealed with asingle sheet of clear adhesive film (such as parcel packaging tape)folded around one edge and sealed at the opposite edge with aface-to-face adhesive seal.

The assembly is inserted into a Perkin-Elmer 480 Thermal Cycler whichhas been modified by milling or sawing a narrow, deep groove across itsface so that the thin microhole chip may be inserted into the groove andthe chip reliably thermal cycled. A thin heat-conductive silicone pad(Stockwell Rubber Company, Philadelphia, Pa.) is used to provide thermalcontact as well as pressure to the surfaces of the microhole chip. Theadded pressure prevents evaporation of the samples. The apparatus isthermal cycled according to the following protocol: (1) 94° C. for 60sec; (2) 60 cycles of 94° C. for 20 sec, 55° C. for 20 sec, 70° C. for20 sec; and (3) 70° C. for 10 minutes.

Following completion of the thermal cycling protocol the chip is scannedusing a two color scanner or fluorescent microscope to determine thelevels of fluorescence due to FAM (6-carboxy-fluorescein) (from exon 35)and TET (tetrachloro-6-carboxy-fluorescein) (from exon 49).

Of the 256 microholes, approximately 142 should have no fluorescentsignal due to lack of DNA template. Of the remaining microholes, a FAMsignal indicates the presence of the IVS34-7 T>C mutation of intron 34and a TET signal indicates the presence of the IVS48-69 insATT mutationof intron 48. The case of a large number of FAM and TET signals from thesame microholes indicates the very high likelihood that both mutationsare present on the same allele. If both FAM and TET appear in the samemicrohole in only a few instances, this more likely indicates thepresence in a single microhole of two different alleles.

Primers and Probes

The following primers are designed to amplify two specific loci onintron 34 and intron 48, separated by approximately 29 kb on the humanATM gene. The probes have been designed to hybridize to the polymorphicregion (as opposed to the wild-type), therefore a FAM signal will begenerated if intron 34 contains the T>C polymorphism and the TET signalwill be generated if intron 48 contains an ATT insertion Primersequences: Intron 34F: caaaagtgttgtcttcatgc Intron 34R:ctgcaacaaattgacaact-agt Intron 48F: taagatagtccctgacaagtagtta Intron48R: tgacatatgggaataaatactttt

Probe Sequences for Two Mutant Alleles of the ATM Gene (Mutations inUpper Case) Intron 34Probe:<fam>tttaaaa<tamra>aattaCttctagataatcc- gcaIntron 48Probe:<tet>ttgctgc<tamra>tttcATT- attattattattcat

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be apparent to those skilled in the art thatvarious changes and modifications can be practiced without departingfrom the spirit of the invention. Therefore the foregoing descriptionsand examples should not be construed as limiting the scope of theinvention.

1. An apparatus for containing multiple micro-volume liquid samplescomprising a substrate, wherein the substrate defines a plurality ofsample chambers, wherein each sample chamber: (a) extends through thesubstrate; (b) comprises one or more walls and an opening at each end;and (c) comprises a hydrophobic annular ring on the wall of the chamber,separating two hydrophilic regions; and (d) holds a sample in the formof a thin film such that a liquid sample present in the sample chamberdoes not intermix with a liquid sample present in another samplechamber.
 2. An apparatus according to claim 1, wherein the samplechamber has a height to width ratio less than or equal to 2:1 when theheight of the sample chamber is measured from one face of the substrateto the other.
 32. 3. An apparatus according to claim 1 wherein thesubstrate comprises hydrophobic regions located on the substrate suchthat a liquid sample present in one sample chamber does not intermixwith a liquid sample present in another sample chamber.
 4. An apparatusaccording to claim 3, wherein the substrate comprises an upper face anda lower face.
 5. An apparatus according to claim 4, wherein the throughaxes of the sample chambers are perpendicular to both faces of thesubstrate.
 6. An apparatus according to claim 5, wherein the samplechamber has the shape of a right circular cylinder.
 7. An apparatusaccording to claim 5, wherein the sample chamber has the shape of aright polygonal prism.
 8. An apparatus according to claim 3, wherein thehydrophobic regions are located on the upper and lower faces of thesubstrate such that the openings of at least one sample chamber isseparated from at least one adjacent sample chamber by a hydrophobicregion.
 9. An apparatus according to claim 8, wherein additionalhydrophobic regions are located on the walls of the sample chambers. 10.An apparatus according to claim 3, wherein the additional hydrophobicregions are located on the walls of the sample chambers.
 11. Anapparatus according to claim 10, wherein the additional hydrophobicregion forms an annular ring along the wall of the sample chamber. 12.An apparatus according to claim 10, comprising two or more hydrophobicregions, each forming an annular ring along the wall of the samplechamber, wherein the hydrophobic regions define one or more annularnon-hydrophobic rings therebetween.
 13. An apparatus according to claim1 further comprising at least one component of a reaction to be carriedout in the apparatus.
 14. An apparatus according to claim 1, wherein areaction component is affixed to the substrate.
 15. An apparatusaccording to claim 13, wherein the component is a reagent used in anucleotide sequencing reaction, a hybridization reaction, or apolynucleotide amplification reaction.
 16. An apparatus according toclaim 1, wherein the apparatus is substantially free from contaminatingamplifiable polynucleotides.
 17. An apparatus according to claim 1,wherein the substrate comprises top and bottom surfaces that eachcontain raised features which form closed curves circumscribing theopenings to said sample chambers.
 18. A kit comprising an apparatus forcontaining multiple micro-volume liquid samples comprising a substrate,wherein the substrate defines a plurality of sample chambers, whereineach sample chamber: (a) extends through the substrate; (b) comprisesone or more walls and an opening at each end; and (c) holds a sample inthe form of a thin film such that a liquid sample present in the samplechamber does not intermix with a liquid sample present in another samplechamber, wherein the kit further includes a reagent used in apolynucleotide amplification reaction.
 19. The kit according to claim18, wherein the sample chamber has a height to width ratio less than 2:1,when the height of the sample chamber is measured from one face of thesubstrate to the other.
 20. A kit according to claim 18, furthercomprising a hydrophobic substance to be used with the apparatus.
 21. Akit according to claim 20, wherein the hydrophobic substance is ahydrophobic fluid packaged in a suitable container.
 22. A kit accordingto claim 20, wherein the hydrophobic substance is a hydrophobic cover.23. A kit according to claim 18, further comprising a chamber formaintaining the appropriate environmental conditions for a reaction tobe carried out in the apparatus.
 24. A kit according to claim 18,further comprising an apparatus for loading samples into the samplechambers.
 25. A kit according to claim 18 comprising a hydrophobiccover.
 26. An apparatus for containing multiple micro-volume liquidsamples comprising a substrate, wherein the substrate defines aplurality of sample chambers, wherein each sample chamber: extendsthrough the substrate; comprises one or more walls and an opening ateach end; and holds a sample in the form of a thin film such that aliquid sample present in the sample chamber does not intermix with aliquid sample present in another sample chamber; and wherein theapparatus includes template, reagent and primer pairs for apolynucleotide amplification reaction.
 27. An apparatus according toclaim 26, wherein the template is DNA.
 28. An apparatus for containingmultiple micro-volume liquid samples comprising a substrate, wherein thesubstrate defines a plurality of sample chambers, wherein each samplechamber: (a) extends through the substrate; (b) comprises one or morewalls and an opening at each end; and (c) holds a sample in the form ofa thin film such that a liquid sample present in the sample chamber doesnot intermix with a liquid sample present in another sample chamber, andfurther comprising a reagent used in a polynucleotide amplificationreaction.
 29. An apparatus of claim 28 wherein the substrate istitanium.
 30. An apparatus of claim 28 wherein the samples chamber havea height to width ration of less than or equal to 2:1 when the height ofthe sample chamber is measured from one face of the substrate to theother.
 31. An apparatus of claim 30 wherein the substrate comprisestitanium.
 32. An apparatus according to claim 28, wherein the chamberscomprise a hydrophobic annular ring on the wall of the chamber,separating two hydrophilic regions.